GSM7977031: Beef steer 7, subcutaneous adipose tissue (SCAT); Bos taurus; RNA-Seq
RNA was extracted from adipose tissue samples using a Qiagen Lipid Minikit (Qiagen, Germantown Road, Germantown, MD; Cat# 74804). In short, 50-75mg of frozen adipose tissue was placed into a polystyrene tube containing two 3mm zirconium beads and 1 mL of Trizol reagent. The tissue was thenhomogenized at 400 m/s in 30s intervals using a bead blaster (Benchmark Scientific Inc, Sayreville, NJ, USA). After homogenization, sample was centrifuged at 12,000 × g for 10 min at 4°C, and a pink middle layer fluid was collected into a new 2-mL RNase-free microtube. Then, 200 µL of Phenol:chloroform (Invitrogen, Carlsbad, CA) at 4°C were added to the sample in order to isolate the RNA from the organic phase. After centrifugation at 12,000 × g for 15 min at 4°C, the clear upper phase was transferred into a new 2-mL RNase-free microtube containing 700 µL of 70% ethanol. The total RNA was purified using RNeasy Plus Mini Kit and eluted in 30 µL of RNase-free water. RNA integirty was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (Rnase H-) using the RNA samples. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. PCR was performed with Phusion High-Fidelity DNA polymerase, Uni-versal PCR primers, and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq 6000 S4 platform to generate 30 million stranded paired-end reads (2 x 150 nt) per sample.